Previous studies have demonstrated that, when the predominantly adrenergic neurons of the neonatal rat superior cervical ganglion (SCG) are grown under certain culture conditions, they acquire many of the properties characteristic of cholinergic neurons. To determine whether this occurs at the expense of certain of their adrenergic properties, cultured SCG neurons were characterized by both biochemical and immunocytochemical methods. We report here data which demonstrate that sympathetic neurons, cultured under conditions which foster the accrual of cholinergic properties, exhibit parallel increases in the activities and amounts of the specific adrenergic enzymes, tyrosine hydroxylase and dopamine beta-hydroxylase, as well as the specific cholinergic enzyme, choline acetyltransferase. Using immunocytochemical methods, we further demonstrate that essentially all SCG neurons stain positively with antibodies to tyrosine hydroxylase, even at times in culture when choline acetyltransferase levels are elevated significantly. These data indicate that virtually all SCG neurons grown in our culture system are capable of dual neurotransmitter production and thus express at least the potential for dual function for up to 7 weeks in culture.